In multicellular animals, cell growth, differentiation, and migration are controlled by polypeptide growth factors and hormones. These growth factors play a role in both normal development and pathogenesis, including the development of solid tumors.
Polypeptide growth factors and hormones influence cellular events by binding to cell-surface receptors. Binding initiates a chain of signalling events within the cell, which ultimately results in phenotypic changes such as cell division and production of additional hormones.
One family of hormones is the glycoprotein hormone family, which includes luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, and chorionic gonadotropin. The first three are synthesized in the anterior pituitary, while chorionic gonadotropin is synthesized in the placenta, reaching a maximum at 10-12 weeks after conception and declining thereafter to the end of pregnancy.
The four glycoprotein hormones are structurally and functionally related. All four are glycosylated and consist of two non-covalently associated subunits, term xcex1 and xcex2 subunits. A single xcex1 subunit is common to all four hormones, while the xcex2 subunits are unique and confer biological specificity. The different xcex2 subunits are of similar size and have a significant degree of pairwise homology; the xcex2 subunits of human chorionic gonadotropin (HCG) and human luteinizing hormone are 82% identical, and the other pairs of xcex2 subunits are about 30-40% identical. Twelve cysteine residues are conserved among the four xcex2 subunits. The common xcex1 subunit exhibits detectable homology to the xcex2 subunits and includes six of the twelve conserved cysteine residues. See, Fiddes and Goodman, Nature 281:351-356, 1979; Fiddes and Goodman, Nature 286:684-687, 1980; Talmadge et al., Nature 307:37-40, 1984; and Pierce and Parsons, Ann. Rev. Biochem. 50:465-495, 1981. The polypeptides form characteristic higher-order structures having a bow tie-like configuration about a cystine knot, formed by disulfide bonding between three pairs of cysteine residues. Dimerization occurs through hydrophobic interactions between loops of the two monomers. See, Daopin et al., Science 257:369, 1992; Lapthorn et al., Nature 369:455, 1994.
The cystine knot motif and bow tie-like fold are also characteristic of the growth factors transforming growth factor-beta (TGF-xcex2), nerve growth factor (NGF), and platelet derived growth factor (PDGF). These proteins are all dimers in their active forms, the monomer subunits of which contain from 100 to 130 amino acid residues. Although their amino acid sequences are quite divergent, these proteins, as well as the glycoprotein hormones, all contain the six conserved cysteine residues of the cystine knot.
The glycoprotein hormones act in a stage- and tissue-specific manner. LH, FSH, and TSH are produced in the pituitary. Luteinizing hormone stimulates steroid production in the testes and ovaries, which in turn stimulates spermatogenesis and ovulation. FSH is also a regulator of gametogenesis and steroid hormone synthesis in the gonads. TSH regulates a variety of processes in the thyroid, thereby controlling synthesis and secretion of thyroid hormones. HCG, produced in placenta, stimulates the ovaries to produce steroids that are necessary for the maintenance of pregnancy. For review see Pierce and Parsons, Ann. Rev. Biochem. 50:465-495, 1981.
A more recently discovered member of this family, designated Norrie disease protein (NDP), is believed to be a regulator of neural cell differentiation and proliferation (Berger et al., Nature Genetics 1:199-203, 1992). NDP is expressed in retina, choroid, and fetal and adult brain. A lack of functional NDP is associated with Norrie disease, an X-linked disorder characterized by blindness, deafness, and mental disturbances. A number of variant forms of the protein, including deletions and point mutations, have been identified in Norrie disease patients. See, for example, Berger et al., ibid.; Fuchs et al., Hum. Mol. Genet. 3:655-656, 1994; and Meindl et al., Nature Genetics 2:139-143, 1992.
Another group of related proteins is the growth and differentiation factors (GDFs). One member of this group, known as GDF-8 or myostatin, appears to act as a negative regulator of muscle mass (McPherron and Lee, Nature 387:83-90, 1997; McPherron and Lee, Proc. Natl. Acad. Sci. USA 94:12457-12461, 1997; and Grobet et al., Nat. Genet. 17:17-71, 1997). Many GDFs share 20-40% sequence homology with each other and with TGF-xcex2 1, 2, and 3. The discovery of the GDFs supports the postulated existence of xe2x80x9cchalonesxe2x80x9d, soluble factors hypothesized to control organ size and regeneration (Bullough, Cancer Res. 25:1683-1727, 1965; Bullough, Biol. Res. 37:307-342, 1992).
The role of hormones in controlling cellular processes makes them likely candidates and targets for therapeutic intervention. Examples of such proteins that are used therapeutically include insulin for the treatment of diabetes and erythropoietin for the treatment of anemia. Gonadotropin has been used to induce ovulation (e.g., Fleming, Am. J. Obstet. Gynecol. 159:376-381, 1988), to induce scrotal descent of cryptorchid testes (Lala et al., J. Urol. 157:1898-1901, 1997), and to stimulate intratesticular testosterone production in men who have undergone varicocelectomy (Yamamoto et al., Arch. Androl. 35:49-55, 1995). Clinical studies have shown that hCG can have antitumor activity against Kaposi""s sarcoma (Gill et al., J. Natl. Cancer Inst. 89:1797-1802, 1997). Assays for the presence of chorionic gonadotropin are used to detect pregnancy. Vaccines against hCG have shown promising results in early tests for preventing pregnancy and inhibiting the growth of hormone-dependent cancers (Talwar, Immunol. Cell Biol. 75:184-189, 1997).
In view of the proven clinical utility of hormones, there is a need in the art for additional such molecules for use as therapeutic agents, diagnostic agents, and research tools and reagents.
The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
Within one aspect of the invention there is provided an isolated polypeptide that is at least 80% identical in amino acid sequence to residues 1 through 106 of SEQ ID NO:2. The polypeptide comprises cysteine residues at positions corresponding to residues 8, 34, 38, 66, 96, and 98 of SEQ ID NO:2, a glycine residue at a position corresponding to residue 36 of SEQ ID NO:2, and beta strands corresponding to residues 9-17, 29-34, 38-43, 59-64, 67-71, and 90-95 of SEQ ID NO:2. Within one embodiment of the invention the isolated polypeptide further comprises cysteine residues at positions corresponding to residues 25, 65, 80, and 101 of SEQ ID NO:2. Within a further embodiment, amino acid residues within the polypeptide at positions corresponding to residues 8, 11, 12, 14, 29, 30, 32, 34, 43, 44, 60, 63, 64, 65, 71, 74, 80, 90, 91, 93, and 94 of SEQ ID NO:2 are Cys, His, Pro, Asn, His, Val, Gln, Cys, Phe, Pro, Thr, Ser, Gln, Cys, Leu, Val, Cys, Ile, Phe, Ala, and Arg, respectively, and an amino acid residue corresponding to residue 75 of SEQ ID NO:2 is Lys or Arg. Within other embodiments the isolated polypeptide comprises residue 1 through residue 106 of SEQ ID NO:2 or residue 1 through residue 106 of SEQ ID NO:29. Within further embodiments, the isolated polypeptide is covalently linked to an affinity tag or to an immunoglobulin constant region.
Within a second aspect of the invention there is provided an isolated protein comprising a first polypeptide complexed to a second polypeptide wherein said protein modulates cell proliferation, differentiation, or metabolism. The first polypeptide is at least 80% identical in amino acid sequence to residues 1 through 106 of SEQ ID NO:2 and comprises cysteine residues at positions corresponding to residues 8, 34, 38, 66, 96, and 98 of SEQ ID NO:2, a glycine residue at a position corresponding to residue 36 of SEQ ID NO:2, and beta strands corresponding to residues 9-17, 29-34, 38-43, 59-64, 67-71, and 90-95 of SEQ ID NO:2. Within one embodiment the first polypeptide further comprises cysteine residues at positions corresponding to residues 25, 65, 80, and 101 of SEQ ID NO:2. Within a further embodiment, amino acid residues of the first polypeptide corresponding to residues 8, 11, 12, 14, 29, 30, 32, 34, 43, 44, 60, 63, 64, 65, 71, 74, 80, 90, 91, 93, and 94 of SEQ ID NO:2 are Cys, His, Pro, Asn, His, Val, Gln, Cys, Phe, Pro, Thr, Ser, Gln, Cys, Leu, Val, Cys, Ile, Phe, Ala, and Arg, respectively; and an amino acid residue corresponding to residue 75 of SEQ ID NO:2 is Lys or Arg. Within another embodiment the protein is a heterodimer. Within a related embodiment the second polypeptide is a glycoprotein hormone common alpha subunit. Within other embodiments the first polypeptide comprises residue 1 through residue 106 of SEQ ID NO:2 or residue 1 through residue 106 of SEQ ID NO:29. Within further embodiments, the protein is a homodimer, such as a homodimer of polypeptides comprising residue 1 through residue 106 of SEQ ID NO:2, or a homodimer of polypeptides comprising residue 1 through residue 106 of SEQ ID NO:29.
Within a third aspect of the invention there is provided an isolated polynucleotide encoding a polypeptide that is at least 90% identical in amino acid sequence to residues 1 through 106 of SEQ ID NO:2, wherein the polypeptide comprises cysteine residues at positions corresponding to residues 8, 34, 38, 66, 96, and 98 of SEQ ID NO:2, a glycine residue at a position corresponding to residue 36 of SEQ ID NO:2, and beta strands corresponding to residues 9-17, 29-34, 38-43, 59-64, 67-71, and 90-95 of SEQ ID NO:2. Within one embodiment the polypeptide further comprises cysteine residues at positions corresponding to residues 25, 65, 80, and 101 of SEQ ID NO:2. Within another embodiment, amino acid residues of the polypeptide corresponding to residues 8, 11, 12, 14, 29, 30, 32, 34, 43, 44, 60, 63, 64, 65, 71, 74, 80, 90, 91, 93, and 94 of SEQ ID NO:2 are Cys, His, Pro, Asn, His, Val, Gln, Cys, Phe, Pro, Thr, Ser, Gln, Cys, Leu, Val, Cys, Ile, Phe, Ala, and Arg, respectively, and an amino acid residue corresponding to residue 75 of SEQ ID NO:2 is Lys or Arg. Within certain additional embodiments of the invention, the polypeptide comprises residue 1 through residue 106 of SEQ ID NO:2 or residue 1 through residue 106 of SEQ ID NO:29. Within another embodiment the polynucleotide further encodes a secretory peptide operably linked to the polypeptide. Within additional embodiments the polynucleotide encodes residue xe2x88x9223 through residue 106 of SEQ ID NO:2 or residue xe2x88x9223 through residue 106 of SEQ ID NO:29. Within further embodiments the polynucleotide comprises a sequence of nucleotides as shown in SEQ ID NO:4 or SEQ ID NO:30 from nucleotide 70 through nucleotide 387. Within other embodiments, the polynucleotide comprises a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 125 through nucleotide 442. Within an additional embodiment the polynucleotide is from 318 to 1000 nucleotides in length. The polynucleotide can be DNA or RNA.
Within a fourth aspect of the invention there is provided an expression vector comprising the following operably linked elements: (a) a transcription promoter; (b) a DNA segment encoding a polypeptide as disclosed above; and (c) a transcription terminator. Within one embodiment the DNA segment further encodes a secretory peptide operably linked to the polypeptide. Within further embodiments the DNA segment encodes residue xe2x88x9223 through residue 106 of SEQ ID NO:2 or residue xe2x88x9223 through residue 106 of SEQ ID NO:29.
Within a fifth aspect of the invention there is provided a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses the polypeptide encoded by the DNA segment. The cell can be used within a method of producing a polypeptide, wherein the method comprises culturing the cell, whereby the cell expresses the polypeptide encoded by the DNA segment, and recovering the expressed polypeptide.
Within a further aspect of the invention there is provided an antibody that specifically binds to an epitope of a polypeptide as disclosed above.
The invention also provides a method for detecting a genetic abnormality in a patient, comprising the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:1 or the complement of SEQ ID NO:1, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; (c) comparing the first reaction product to a control reaction product, wherein a difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient.
The invention also provides an oligonucleotide probe or primer comprising 14 contiguous nucleotides of a polynucleotide of SEQ ID NO:4 or a sequence complementary to SEQ ID NO:4. Within one embodiment the probe or primer comprises 14 contiguous nucleotides of a polynucleotide of SEQ ID NO:1 or a sequence complementary to SEQ ID NO:1.
The invention also provides a pharmaceutical composition comprising a polypeptide as disclosed above in combination with a pharmaceutically acceptable vehicle.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and the attached drawing.